Journal: Glia
Article Title: Haploinsufficiency of microglial MyD88 ameliorates Alzheimer's pathology and vascular disorders in APP/PS1-transgenic mice.
doi: 10.1002/glia.24007
Figure Lengend Snippet: FIGURE 6 Haploinsufficiency of microglial MyD88 increases LRP1 in cerebral micro-vessels of APP/PS1-transgenic mice and IL-1β treatment decreases LRP1 in cultured pericytes. Micro blood vessels were isolated from the brains of 9-month-old APPtgMyD88fl/wtCre+/−(MyD88 het) and APPtgMyD88fl/wtCre−/−(MyD88 wt) mice, which were injected with tamoxifen 3 months ago. Protein levels of LRP1, ABCB1, PDGFRβ, and CD13 were determined with quantitative Western blot (a–e). Haploinsufficiency of MyD88 in microglia significantly elevates LRP1 protein level but not for other proteins tested, compared with MyD88-wildtype APP/PS1-transgenic mice (b; t test, n ≥6 per group). Immortalized pericytes from human cerebral capillaries were cultured and treated with IL-1β at various concentrations for 24 hr (f–i) or for 8 days with and without withdrawal of IL-1β during the last 3 days (j–m). At the end of experiments, cell lysates were prepared from IL-1β-treated pericytes and detected for LRP1, PDGFRβ, and CD13 with quantitative Western blot. One-way ANOVA comparing levels of each tested protein at different concentrations of IL-1β shows that: (1) IL-1β treatments significantly decreases LRP1, but increases PDGFRβ and CD13 in a concentration-dependent manner after a 24-hr treatment (g–i; p values are shown in the figure); (2) IL-1β treatments significantly decreases LRP1 in a concentration-dependent manner after a 8-day treatment (k; p <.001); and (3) IL-1β treatments does not significantly changes protein levels of PDGFRβ and CD13 after a 8-day treatment (l,m). Two-way ANOVA comparing protein levels of LRP1, PDGFRβ, or CD13 with and without withdrawal of IL-1β in the last 3 days of experiments shows that withdraw of IL-1β recovers expression of LRP1 (k; p = .004), but not for PDGFRβ and CD13 (l,m). t test was used to analyze the difference of protein levels in cells treated with IL- 1β at 50 ng/ml shows that withdrawal of IL-1β significantly recovers expression of LRP1, PDGFRβ, and CD13 (k–m; **: p <.01). n = 3 or 4 per group [Color figure can be viewed at wileyonlinelibrary.com]
Article Snippet: For the detection of proteins in cerebral capillaries, rabbit monoclonal antibodies against platelet-derived growth factor receptor β (PDGFRβ), CD13/APN, ABCB1, and vinculin (clone: 28E1, D6V1W, E1Y7S, and E1E9V respec- tively; Cell Signaling Technology) and rabbit polyclonal antibodies against LRP1 and α-tubulin (Catalog numbers: 64099 and 2144, respectively; Cell Signaling Technology), tight junction protein 1 (TJP1; Catalog num- bers: NBP1-85047; Novus Biologicals, Wiesbaden-Nordenstadt, Ger- many), Claudin-5 (Thermo Fisher Scientific) and aquaporin 4 (AQP4; Proteintech Europe, Manchester, United Kingdom) were used.
Techniques: Transgenic Assay, Cell Culture, Isolation, Injection, Western Blot, Concentration Assay, Expressing
Proteintech is a verified supplier 
![FIGURE 6 Haploinsufficiency of microglial MyD88 increases <t>LRP1</t> in cerebral micro-vessels of APP/PS1-transgenic mice and IL-1β treatment decreases LRP1 in cultured pericytes. Micro blood vessels were isolated from the brains of 9-month-old APPtgMyD88fl/wtCre+/−(MyD88 het) and APPtgMyD88fl/wtCre−/−(MyD88 wt) mice, which were injected with tamoxifen 3 months ago. Protein levels of LRP1, ABCB1, PDGFRβ, and CD13 were determined with quantitative Western blot (a–e). Haploinsufficiency of MyD88 in microglia significantly elevates LRP1 protein level but not for other proteins tested, compared with MyD88-wildtype APP/PS1-transgenic mice (b; t test, n ≥6 per group). Immortalized pericytes from human cerebral capillaries were cultured and treated with IL-1β at various concentrations for 24 hr (f–i) or for 8 days with and without withdrawal of IL-1β during the last 3 days (j–m). At the end of experiments, cell lysates were prepared from IL-1β-treated pericytes and detected for LRP1, PDGFRβ, and CD13 with quantitative Western blot. One-way ANOVA comparing levels of each tested protein at different concentrations of IL-1β shows that: (1) IL-1β treatments significantly decreases LRP1, but increases PDGFRβ and CD13 in a concentration-dependent manner after a 24-hr treatment (g–i; p values are shown in the figure); (2) IL-1β treatments significantly decreases LRP1 in a concentration-dependent manner after a 8-day treatment (k; p <.001); and (3) IL-1β treatments does not significantly changes protein levels of PDGFRβ and CD13 after a 8-day treatment (l,m). Two-way ANOVA comparing protein levels of LRP1, PDGFRβ, or CD13 with and without withdrawal of IL-1β in the last 3 days of experiments shows that withdraw of IL-1β recovers expression of LRP1 (k; p = .004), but not for PDGFRβ and CD13 (l,m). t test was used to analyze the difference of protein levels in cells treated with IL- 1β at 50 ng/ml shows that withdrawal of IL-1β significantly recovers expression of LRP1, PDGFRβ, and CD13 (k–m; **: p <.01). n = 3 or 4 per group [Color figure can be viewed at wileyonlinelibrary.com]](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_4399/pm33934399/pm33934399__page11_image1.jpg)
